# The Morphogenesis of Sperm Storage Micropockets in the Western Mosquitofish (Gambusia affinis)

## TL;DR

This paper explains how sperm storage micropockets form in the western mosquitofish, revealing a consistent process involving protrusions from developing eggs.

## Contribution

The study identifies a universal morphogenetic process for sperm storage micropockets in Poeciliidae fish, driven by protrusions from previtellogenic oocytes.

## Key findings

- Protrusions from previtellogenic oocytes form and break off, creating sac-like structures that become sperm storage micropockets.
- Similar protrusions are observed across multiple Poeciliidae species, suggesting a conserved developmental mechanism.
- The resulting micropocket is lined by a single layer of germinal epithelium, suitable for long-term sperm storage.

## Abstract

The females of species undergoing internal fertilization often possess specialized structures or organs in their reproductive system to store spermatozoa. Here, we investigated the formation of sperm storage structures in the western mosquitofish (Gambusia affinis), and we describe how a sperm storage micropocket (SSP) begins. This process is initiated when a protrusion forms on a previtellogenic oocyte, a feature that exhibits consistency in other poeciliid fishes. This protrusion grows, pushes through the outer layer of tissue, and breaks off into the ovarian lumen (we have also observed the emergence of similar protrusion structures in other poeciliid fishes). The sac-like space formed by the compression of the germinal epithelium due to the protrusion represents the initial formation of the SSP, after which a single layer of germinal epithelium forms at the base, developing into a mature SSP. This study reveals the reason for the formation of the SSP and its morphogenesis.

Sperm storage within the female reproductive tract is widespread among animal species with internal fertilization, such as the fish family Poeciliidae, wherein the females possess a sperm storage micropocket (SSP) that can store sperm for up to a year. However, the underlying mechanism and morphogenesis of SSP remain unclear. Here, using histological analysis, we found that the SSP of the western mosquitofish (Gambusia affinis) was associated with protrusions arising from the previtellogenic oocyte (PO). Similar protrusions occur in the PO of Poecilia reticulata, P. latipinna, Xiphophorus helleri, and X. maculatus, suggesting that such PO-derived protrusions might be a universal feature in Poeciliidae. These protrusions exhibit significant differences in structural composition compared to the cytoplasm. Furthermore, the area of these protrusions positively correlates with the total area of the oocytes. After maturing, the protrusions split from the oocytes, leaving behind sac-like structures composed of germinal epithelium, which later develop into the SSP for storing sperm. The bottom of the SSP is composed of a single layer of germinal epithelium. We describe and depict the protrusion formation process in the fish family Poeciliidae during the previtellogenic stage, elucidating the SSP morphogenesis.